At least three major, noninterconvertible isozymes of pyruvate kinase (EC 2.7.1.40) have been identified in mammalian tissues. The dominant species in normal adult liver is designated type L, while the two muscle type isozymes are designated M1 and M2. Fetal and hepatoma tissue contain mainly the M2 isozyme. Both M1 and M2 are found in the brain, where their inhibition by phenylalanine is thought to contribute to the mental retardation associated with phenylketonuria. Some reports claim that erythrocytes contain a fourth isozyme, although this enzyme may also be identical to the type L isozyme. Deficiencies in erythrocyte pyruvate kinase are associated with an important class of hereditary, nonspherocytic hemolytic anemias. We hope to settle the identity of the erythrocyte isozyme and to resolve several other ambiguities regarding the distribution of mammalian pyruvate kinases by means of electrophoretic analysis of extracts from tissues and from preparations of specific cell types, by comparative studies on purified enzymes, and by immunofluorescent techniques. Kinetic and other physicochemical studies will also be performed on hybrid isozymes of pyruvate kinase. M2-M1 and M2-L hybrid sets exist in vivo; the latter set has also been produced by others in vitro. Although the third hybrid pairing, M1 with L, does not exist in vivo, we have performed the hybridization artificially and have isolated each of the products. Because the L isozyme is an allosteric enzyme while M1 exhibits only hyperbolic kinetics with its substrates, a further examination of these hybrid enzymes should provide important information concerning the relationship between subunit interactions and regulatory properties. Through characterization of the major pyruvate kinase isozymes and their hybrids, we hope to better define the roles of the different isozymes in normal carbohydrate metabolism as well as in clinically important abnormal conditions.